Project Summary/Abstract The cytosolic iron sulfur (FeS) cluster assembly (CIA) pathway is essential since it supplies FeS clusters to enzymes which are essential for DNA replication and repair, transcription, and translation. Despite CIA's unquestionable importance for cell growth and division, we understand little about its mechanism. In particular, there is a dearth of information regarding how the >20 FeS-enzyme ?targets? differing in their sequence, structure and function are all identified as CIA substrates so that their cofactors can be inserted in the final step of the pathway. Recent work has pinpointed a multiprotein complex, termed the CIA targeting complex (CTC), as being essential for this apo-target recognition step. The CTC subunits, called Met18, Cia1 and Cia2 in yeast, are highly conserved across the eukaryotic kingdom and their depletion results in a defect in FeS cofactor maturation in cytosolic and nuclear, but not mitochondrial, enzymes. However, progress to understand the final step of CIA has been slow due, in part, to the inability to access a reconstituted in vitro system for mechanistic analysis. We have recently overcome this barrier and are now poised to begin elucidating the mechanism by which apo-targets are identified. The remarkable ability of the CTC to flexibly yet specifically recognize the diverse pool of CIA targets is likely accomplished by Met18, Cia1 and Cia2 forming multiple distinct complexes, each responsible for recognition of a distinct subset of targets, by individual targets or subsets of targets sharing a common targeting motif sufficient for association with the CTC or by a combination of these two mechanisms. To reveal the molecular details underlying apo-target identification, we will 1) identify residues critical for formation of the targeting complex and evaluate how their mutation affects CIA function in vivo; 2) complete a comprehensive screen to identify functional residues of CTC subunits and pinpoint which are essential for target-binding in vitro and in vivo; and 3) elucidate the targeting motif exploited by the cytosolic FeS protein Leu1 and the nuclear FeS protein Rad3 for their association with the targeting complex. Successful completion of these aims is expected to provide fundamental knowledge about the structure of the targeting complex and the role(s) of its individual subunits, reveal the cryptic code driving CIA target recognition, and yield new information about how different targets or subsets of targets are identified by the CIA system. Since target recognition is not well understood for any cluster biogenesis pathway, this project is expected to shed light on this long-standing black box in the cluster biogenesis field. Finally, since defects CIA function result in sensitivity to DNA damaging agents, chromosomal instabilities, elongated telomeres and other genotoxic phenotypes, elucidation of the fundamental mechanism underlying CIA substrate recognition is expected to provide information essential for understanding the relationship between CIA and genome stability and the impact of CIA deficiencies on human health and disease.